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Abstract:

The present invention provides methods for improving treatment effect in
a patient suffering from gastric cancer, in particular, adenocarcinoma of
the stomach or gastro-esophageal junction ("GEJ"), by treatment with
bevacizumab (Avastin®) in combination with a chemotherapy regimen by
determining the expression level of neuropilin relative to a control
level determined in patients suffering from gastric cancer, in
particular, adenocarcinoma of the stomach or gastro-esophageal junction
("GEJ"). The improved treatment effect may be improved overall survival
or improved progression free survival. The present invention further
provides for methods for assessing the sensitivity or responsiveness of a
patient to bevacizumab (Avastin®) in combination with a chemotherapy
regimen, by determining the expression level of neuropilin relative to a
control level determined in patients suffering from gastric cancer, in
particular, adenocarcinoma of the stomach or gastro-esophageal junction
("GEJ").

Claims:

1. A method of selecting a cancer treatment for a patient suffering from
gastric cancer, said method comprising: (a) determining that a sample
obtained from the patient has a decreased expression level of neuropilin
relative to a control level determined in patients suffering from gastric
cancer; and (b) providing a recommendation that said cancer treatment
selected for the patient comprise an effective amount of bevacizumab in
combination with a chemotherapy regimen.

2. A method of monitoring patient response to a cancer treatment for a
patient suffering from gastric cancer, said method comprising: (a)
determining that a sample obtained from the patient has a decreased
expression level of neuropilin relative to a control level determined in
patients suffering from gastric cancer; and (b) providing a
recommendation that said cancer treatment for the patient comprise an
effective amount of bevacizumab in combination with a chemotherapy
regimen.

3. A method for the identification of a patient responsive to, or
sensitive to, the addition of bevacizumab treatment to a chemotherapy
regimen, said method comprising: determining an expression level of
neuropilin in a patient sample from a patient suspected to suffer from or
being prone to suffer from gastric cancer, said patient sample being
contacted with EDTA, whereby a decreased level of neuropilin relative to
a control level determined in patients suffering from gastric cancer
identifies the patient as being responsive to or sensitive to the
addition of bevacizumab to said chemotherapy regimen.

4. A method of predicting the response to or sensitivity to the addition
of bevacizumab to a chemotherapy regimen of a patient suspected to suffer
from, suffering from or prone to suffer from gastric cancer, said method
comprising: determining an expression level of neuropilin in a patient
sample from a patient suspected to suffer from or being prone to suffer
from gastric cancer, said patient sample being contacted with EDTA,
whereby a decreased level of neuropilin relative to a control level
determined in patients suffering from gastric cancer predicts the
response to or sensitivity to the addition of bevacizumab to said
chemotherapy regimen.

5. The method of claim 3 or 4, further comprising informing the patient
that they may benefit from cancer treatment comprising an effective
amount of bevacizumab in combination with a chemotherapy regimen.

6. The method of any one of claims 1 to 4, wherein said gastric cancer is
stomach adenocarcinoma or gastroesophageal junction adenocarcinoma.

7. The method of claim 1, wherein said chemotherapy regimen is a
capecitabine-based chemotherapy regimen or a 5-fluorouracil-based
chemotherapy regimen.

8. The method of claim 7, wherein said capecitabine-based chemotherapy
regimen is a regimen of capecitabine in combination with cisplatin.

9. The method of claim 7, wherein said 5-fluorouracil-based chemotherapy
regimen is a regimen of 5-fluorouracil in combination with cisplatin.

10. The method of claim 1, wherein the patient sample is selected from
the group consisting of: whole blood, plasma, serum, and combinations
thereof.

11. The method of claim 1, wherein said patient sample is selected from
the group consisting of gastric tissue resection or gastric tissue
biopsy.

12. The method of claim 1, wherein the expression level is a protein
expression level.

14. The method of claim 12, wherein a plasma level of neuropilin in a
sample obtained from the patient that is at or below the level of
neuropilin in a reference sample, indicates that the patient may benefit
from the addition of bevacizumab treatment to said chemotherapy regimen,
or has increased likelihood of benefit from the addition of bevacizumab
treatment to said chemotherapy regimen.

15. The method of claim 1, wherein said neuropilin expression level is
detected by an immunohistochemical method (IHC).

16. The method of claim 1, further comprising administering a
therapeutically effective amount of bevacizumab in combination with a
chemotherapy to the patient having a decreased level of neuropilin
relative to a control level determined in patients suffering from gastric
cancer.

17. The method of claim 16, wherein said patient is being co-treated with
one or more anti-cancer therapies.

18. The method of claim 17, wherein said anti-cancer therapy is
radiation.

19. The method of claim 1, wherein the expression level of neuropilin is
determined before neoadjuvant or adjuvant therapy.

Description:

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. application Ser. No.
13/737,586, filed Jan. 9, 2013, which is a continuation of international
application PCT/EP2011/063932, filed Aug. 12, 2011 which claims priority
from European Patent Application 10172812.9, filed Aug. 13, 2010, the
contents of which are incorporated herein by reference.

[0002] The present invention provides methods for improving treatment
effect in a patient suffering from gastric cancer, in particular,
adenocarcinoma of the stomach or gastro-esophageal junction ("GEJ"), by
treatment with bevacizumab (Avastin®) in combination with a
chemotherapy regimen by determining the expression level of neuropilin
relative to a control level determined in patients suffering from gastric
cancer, in particular, adenocarcinoma of the stomach or gastro-esophageal
junction ("GEJ"). The improved treatment effect may be improved overall
survival or improved progression free survival. The present invention
further provides for methods for assessing the sensitivity or
responsiveness of a patient to bevacizumab (Avastin®) in combination
with a chemotherapy regimen, by determining the expression level of
neuropilin relative to a control level determined in patients suffering
from gastric cancer, in particular, adenocarcinoma of the stomach or
gastro-esophageal junction ("GEJ").

[0003] Accordingly, the present invention relates to the identification
and selection of one or more biomarkers of gastric cancer, in particular,
adenocarcinoma of the stomach or gastro-esophageal junction ("GEJ"), that
correlate with sensitivity or responsiveness to angiogenesis inhibitors,
e.g., bevacizumab (Avastin®), in combination with chemotherapeutic
regimens, such as capecitabine- or 5-fluorouracil-based chemotherapies.
In certain aspects, the invention relates to the use of the tumor
specific expression of neuropilin determined relative to controls
established in patients suffering from gastric cancer, in particular,
adenocarcinoma of the stomach or GEJ, to identify patients sensitive or
responsive to the addition of angiogenesis inhibitors, e.g., bevacizumab
(Avastin®), to standard chemotherapies. The invention also relates to
methods for improving treatment effect in a patient suffering from
gastric cancer, in particular, adenocarcinoma of the stomach or GEJ, by
the addition of angiogenesis inhibitors, e.g., bevacizumab
(Avastin®), to standard chemotherapies, e.g., capecitabine- or
5-fluorouracil-based chemotherapies, by determining the tumor specific
expression level of neuropilin relative to a control established in
patients suffering from gastric cancer, in particular, adenocarcinoma of
the stomach or GEJ. Treatment effect includes the clinical parameters
overall survival and progression free survival. The invention also
provides for kits and compositions for identification of patients
sensitive or responsive to angiogenesis inhibitors, in particular,
bevacizumab (Avastin®), which patients are determined and defined in
accordance with the methods described herein.

[0004] Angiogenesis is necessary for cancer development, regulating not
only primary tumor size and growth but also impacting invasive and
metastatic potential. Accordingly, the mechanisms mediating angiogenic
processes have been investigated as potential targets for directed
anti-cancer therapies. Early in the study of angiogenic modulators, the
vascular endothelial growth factor (VEGF) signalling pathway was
discovered to preferentially regulate angiogenic activity in multiple
cancer types and multiple therapeutics have been developed to modulate
this pathway at various points. These therapies include, among others,
bevacizumab, sunitinib, sorafenib and vatalanib. Although the use of
angiogenic inhibitors in the clinic has shown success, not all patients
respond or fail to fully respond to angiogenesis inhibitor therapy. The
mechanism(s) underlying such incomplete response is unknown. Therefore,
there is an increasing need for the identification of patient subgroups
sensitive or responsive to anti-angiogenic cancer therapy.

[0005] While a number of angiogenesis inhibitors are known, the most
prominent angiogenesis inhibitor is Bevacizumab (Avastin®).
Bevacizumab is a recombinant humanized monoclonal IgG1 antibody that
specifically binds and blocks the biological effects of VEGF (vascular
endothelial growth factor). VEGF is a key driver of tumor
angiogenesis--an essential process required for tumor growth and
metastasis, i.e., the dissemination of the tumor to other parts of the
body. Avastin® is approved in Europe for the treatment of the
advanced stages of four common types of cancer: colorectal cancer, breast
cancer, non-small cell lung cancer (NSCLC) and kidney cancer, which
collectively cause over 2.5 million deaths each year. Over half a million
patients have been treated with Avastin® so far, and a comprehensive
clinical program with over 450 clinical trials is investigating the
further use of Avastin in the treatment of multiple cancer types
(including colorectal, breast, non-small cell lung, brain, gastric,
ovarian and prostate) in different settings (e.g., advanced or early
stage disease). Importantly, Avastin® has shown promise as a
co-therapeutic, demonstrating efficacy when combined with a broad range
of chemotherapies and other anti-cancer treatments. Phase-III studies
have been published demonstrating the beneficial effects of combining
bevacizumab with standard chemotherapeutic regimens (see, e.g., Kang et
al., 2010, J. Clin. Oncol., 28:18s (suppl. abstr. LBA4007); Saltz et al.,
2008, J. Clin. Oncol., 26:2013-2019; Yang et al., 2008, Clin. Cancer
Res., 14:5893-5899; Hurwitz et al., 2004, N Engl. J. Med.,
350:2335-2342). However, as in previous studies of angiogenic inhibitors,
some of these phase-III studies have shown that a portion of patients
experience incomplete response to the addition of bevacizumab
(Avastin®) to their chemotherapeutic regimens.

[0006] Accordingly, there is a need for methods of determining those
patients that respond or are likely to respond to combination therapies
comprising angiogenesis inhibitors, in particular, bevacizumab
(Avastin®). Thus, the technical problem underlying the present
invention is the provision of methods and means for the identification of
(a) patient(s) suffering from or prone to suffer from gastric cancer, in
particular, adenocarcinoma of the stomach or GEJ, who may benefit from
the addition of angiogenesis inhibitors, in particular, bevacizumab
(Avastin®), to chemotherapeutic regimens, e.g., capecitabine- or
5-fluorouracil-based chemotherapies.

[0007] The technical problem is solved by provision of the embodiments
characterized in the claims.

[0008] The present invention, therefore, provides a method for improving
treatment effect in a patient suffering from gastric cancer, in
particular, adenocarcinoma of the stomach or GEJ, by adding bevacizumab
to a chemotherapy regimen, said method comprising:

[0009] (a)
determining the expression level of neuropilin in a patient sample; and

[0010] (b) administering bevacizumab in combination with a chemotherapy
regimen to the patient having a decreased level of neuropilin relative to
a control level determined in patients suffering from gastric cancer, in
particular, adenocarcinoma of the stomach or GEJ. The improved treatment
effect may be the clinical parameter overall survival or may be
progression free survival.

[0011] In other embodiments, the present invention relates to an in vitro
method for the identification of a patient responsive to or sensitive to
the addition of bevacizumab to a chemotherapy regimen, said method
comprising determining the expression level of neuropilin in a sample
from a patient suspected to suffer from or being prone to suffer from
gastric cancer, in particular, adenocarcinoma of the stomach or GEJ,
whereby decreased level of neuropilin relative to a control level
determined in patients suffering from gastric cancer, in particular,
adenocarcinoma of the stomach or GEJ, is indicative of a sensitivity of
the patient to the addition of bevacizumab to said regimen.

[0012] Accordingly, the present invention solves the identified technical
problem in that it was surprisingly shown that the tumor specific
expression level of neuropilin in a given patient, relative to a control
level determined in patients diagnosed with gastric cancer, in
particular, adenocarcinoma of the stomach or GEJ, correlates with
treatment effect in those patients administered an angiogenesis inhibitor
in combination with a chemotherapy regimen. Variation in the tumor
specific expression level of neuropilin was surprisingly identified as a
marker/predictor for the improved progression-free survival and/or
improved overall survival of gastric cancer patients in response to the
addition of bevacizumab (Avastin®) to capecitabine- or
5-fluorouracil-based chemotherapeutic regimens. Specifically, gastric
cancer patients exhibiting a response or sensitivity to the addition of
bevacizumab (Avastin®) to chemotherapy regimens were identified to
have decreased expression of neuropilin relative to a control level
established in samples obtained from patients suffering from or diagnosed
with gastric cancer, in particular, adenocarcinoma of the stomach or GEJ.
The terms "marker" and "predictor" can be used interchangeably and refer
to the expression level of neuropilin as described and defined herein.

[0013] In the context of the present invention, "neuropilin" refers to the
neuropilin-1 protein, a type-I membrane protein also known as NRP-1, and
exemplified by the amino acid sequence SEQ ID NO:1, shown in FIG. 3 (The
NRP-1 precursor amino acid sequence is also available under UniProt
accession number 014786). As used herein, "neuropilin" may also refer to
neuropilin-2 (also known as NRP-2), which shares approximately 44%
homology to NRP-1 as known in the art. Accordingly, the methods of the
invention do not distinguish between NRP-1 and NRP-2. In the context of
the present invention, the term "neuropilin" also encompasses homologs,
variants and isoforms of NRP-1 and/or NRP-2, so long as said homologs,
variants and isoforms are specifically recognized by one or more
anti-neuropilin antibodies as described herein and/or as known in the
art. The term, "neuropilin" further encompasses proteins having at least
85%, at least 90% or at least 95% homology to the amino acid sequence of
SEQ ID NO:1, or to the sequence of one or more of a NRP-1 and/or NRP-2
homologue, variant and isoform, including splice isoforms, as well as
fragments of the sequences, provided that the variant proteins (including
isoforms), homologous proteins and/or fragments are recognized by one or
more NRP-1 and/or NRP-2 specific antibodies, such as clone 446915
available from R&D Systems, Inc. (Minneapolis, Minn., U.S.A.), that
available as catalog number sc-5307 from Santa Cruz Biotechnology, Inc.
(Santa Cruz, Calif., U.S.A.) or that are otherwise known in the art.

[0014] Accordingly, the present invention encompasses the determination of
expression levels of proteins including, but not limited to, the amino
acid sequences as described herein. In certain aspects, the invention
encompasses the detection of homologues, variants and isoforms of
neuropilin; said isoforms or variants may, inter alia, comprise allelic
variants or splice variants. Also envisaged is the detection of proteins
that are homologous to neuropilin as herein described, or a fragment
thereof, e.g., having at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or
99% sequence identity to the amino acid sequence of SEQ ID NO:1 or a
fragment thereof. Alternatively or additionally, the present invention
encompasses detection of the expression levels of proteins encoded by
nucleic acid sequences, or fragments thereof, that are at least at least
60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleic acid
sequence encoding SEQ ID NO:1 or a fragment, variant or isoform thereof.
In this context, the term "variant" means that the neuropilin amino acid
sequence, or the nucleic acid sequence encoding said amino acid sequence,
differs from the distinct sequences identified by SEQ ID NO:1 and/or
available under the above-identified UniProt Accession numbers, by
mutations, e.g., deletion, additions, substitutions, inversions etc. In
addition, the term "homologue" references molecules having at least 60%,
more preferably at least 80% and most preferably at least 90% sequence
identity to one or more of the polypeptides as shown in SEQ ID NOs:1 or
(a) fragment(s) thereof.

[0015] In order to determine whether an amino acid or nucleic acid
sequence has a certain degree of identity to an amino acid or nucleic
acid sequence as herein described, the skilled person can use means and
methods well known in the art, e.g. alignments, either manually or by
using computer programs known in the art or described herein.

[0016] In accordance with the present invention, the term "identical" or
"percent identity" in the context of two or more or amino acid or nucleic
acid sequences, refers to two or more sequences or subsequences that are
the same, or that have a specified percentage of amino acid residues or
nucleotides that are the same (e.g., 60% or 65% identity, preferably,
70-95% identity, more preferably at least 95% identity with the amino
acid sequences of, e.g., SEQ ID NO:1), when compared and aligned for
maximum correspondence over a window of comparison, or over a designated
region as measured using a sequence comparison algorithm as known in the
art, or by manual alignment and visual inspection. Sequences having, for
example, 60% to 95% or greater sequence identity are considered to be
substantially identical. Such a definition also applies to the complement
of a test sequence. Preferably the described identity exists over a
region that is at least about 15 to 25 amino acids or nucleotides in
length, more preferably, over a region that is about 50 to 100 amino
acids or nucleotides in length. Those having skill in the art will know
how to determine percent identity between/among sequences using, for
example, algorithms such as those based on CLUSTALW computer program
(Thompson Nucl. Acids Res. 2 (1994), 4673-4680) or FASTDB (Brutlag Comp.
App. Biosci. 6 (1990), 237-245), as known in the art.

[0017] Although the FASTDB algorithm typically does not consider internal
non-matching deletions or additions in sequences, i.e., gaps, in its
calculation, this can be corrected manually to avoid an overestimation of
the % identity. CLUSTALW, however, does take sequence gaps into account
in its identity calculations. Also available to those having skill in
this art are the BLAST (Basic Local Alignment Search Tool) and BLAST 2.0
algorithms (Altschul, 1997, Nucl. Acids Res. 25:3389-3402; Altschul, 1993
J. Mol. Evol. 36:290-300; Altschul, 1990, J. Mol. Biol. 215:403-410). The
BLASTN program for nucleic acid sequences uses as defaults a word length
(W) of 11, an expectation (E) of 10, M=5, N=4, and a comparison of both
strands. For amino acid sequences, the BLASTP program uses as defaults a
wordlength (W) of 3, and an expectation (E) of 10. The BLOSUM62 scoring
matrix (Henikoff (1989) PNAS 89:10915) uses alignments (B) of 50,
expectation (E) of 10, M=5, N=4, and a comparison of both strands.

[0018] BLAST algorithms, as discussed above, produce alignments of both
amino and nucleotide sequences to determine sequence similarity. Because
of the local nature of the alignments, BLAST is especially useful in
determining exact matches or in identifying similar sequences. The
fundamental unit of BLAST algorithm output is the High-scoring Segment
Pair (HSP). An HSP consists of two sequence fragments of arbitrary but
equal lengths whose alignment is locally maximal and for which the
alignment score meets or exceeds a threshold or cut-off score set by the
user. The BLAST approach is to look for HSPs between a query sequence and
a database sequence, to evaluate the statistical significance of any
matches found, and to report only those matches which satisfy the
user-selected threshold of significance. The parameter E establishes the
statistically significant threshold for reporting database sequence
matches. E is interpreted as the upper bound of the expected frequency of
chance occurrence of an HSP (or set of HSPs) within the context of the
entire database search. Any database sequence whose match satisfies E is
reported in the program output.

[0019] Analogous computer techniques using BLAST may be used to search for
identical or related molecules in protein or nucleotide databases such as
GenBank or EMBL. This analysis is much faster than multiple
membrane-based hybridizations. In addition, the sensitivity of the
computer search can be modified to determine whether any particular match
is categorized as exact or similar. The basis of the search is the
product score which is defined as:

% sequence identity×% maximum BLAST score/100

and takes into account both the degree of similarity between two
sequences and the length of the sequence match. For example, with a
product score of 40, the match will be exact within a 1-2% error; and at
70, the match will be exact. Similar molecules are usually identified by
selecting those which show product scores between 15 and 40, although
lower scores may identify related molecules. Another example for a
program capable of generating sequence alignments is the CLUSTALW
computer program (Thompson, 1994, Nucl. Acids Res. 2:4673-4680) or FASTDB
(Brutlag, 1990, Comp. App. Biosci. 6:237-245), as is known in the art.

[0021] The expression level of neuropilin (e.g., NRP-1, NRP-2, or a
variant, homologue, truncation or fragment thereof) may be assessed by
any method known in the art suitable for determination of specific
protein levels in a patient sample and is preferably determined by an
immunohistochemical ("IHC") method employing antibodies specific for
neuropilin. Such methods are well known and routinely implemented in the
art and corresponding commercial antibodies and/or kits are readily
available. For example, commercially available antibodies specific for
neuropilin as described and defined herein can be obtained from R&D
Systems, Inc. (Minneapolis, Minn., U.S.A.) as clone 446915 and from Santa
Cruz Biotechnology, Inc. (Santa Cruz, Calif., U.S.A.) as catalog number
sc-5307. Preferably, the expression levels of the marker/indicator
proteins of the invention are assessed using the reagents and/or protocol
recommendations of the antibody or kit manufacturer. The skilled person
will also be aware of further means for determining the expression level
of neuropilin by IHC methods. Therefore, the expression level of
neuropilin and/or other markers/indicators as known in the art can be
routinely and reproducibly determined by a person skilled in the art
without undue burden. However, to ensure accurate and reproducible
results, the invention also encompasses the testing of patient samples in
a specialized laboratory that can ensure the validation of testing
procedures.

[0022] Preferably, the expression level of neuropilin is assessed in a
biological sample that contains or is suspected to contain cancer cells
and is determined in a tumor-specific manner. The sample may comprise
both cancer cells, i.e., tumor cells, and non-cancerous cells, e.g.,
endothelial or non-malignant cells. In some aspects, determination of the
tumor-specific expression of neuropilin relates to the determination of
the expression levels of exclusively cancer cells as opposed to other
cell types, e.g., endothelial or non-cancerous/non-malignant cells, that
may be present in the tumor sample. In other aspects, determination of
the tumor-specific expression of neuropilin relates to the determination
of expression levels of cancer cells as well as any other cell-type,
e.g., endothelial cells, that may be present in the tumor sample. The
skilled artisan, e.g., a pathologist, can readily discern cancer cells
from non-cancerous cells, e.g., endothelial cells. The sample may be a
gastric tissue resection or a gastric tissue biopsy obtained from a
patient suffering from, suspected to suffer from or diagnosed with
gastric cancer, in particular, adenocarcinoma of the stomach or GEJ. The
sample may also be a resection or biopsy of a metastatic lesion obtained
from a patient suffering from, suspected to suffer from or diagnosed with
gastric cancer, in particular, adenocarcinoma of the stomach or GEJ.
Preferably, the sample is a sample of stomach tissue or tissue of the
gastro-esophageal junction, or a resection or biopsy of an adenocarcinoma
of the stomach or gastro-esophageal junction. The sample may also be a
sample of a known or suspected metastatic gastric cancer lesion or
section, or a blood sample, e.g., a peripheral blood sample, known or
suspected to comprise circulating cancer cells, e.g., gastric cancer
cells. The analysis of the sample according to the methods of the
invention may be manual, as performed by the skilled artisan, e.g., a
pathologist, as is known in the art, or may be automated using
commercially available software designed for the processing and analysis
of pathology images, e.g., for analysis in tissue biopsies or resections
(e.g., MIRAX SCAN, Carl Zeiss AG, Jena, Germany). Methods of obtaining
biological samples including tissue resections, biopsies and body fluids,
e.g., blood samples comprising cancer/tumor cells, are well known in the
art.

[0023] In the context of the present invention, bevacizumab is to be
administered in addition to or as a co-therapy or co-treatment with one
or more chemotherapeutic agents administered as part of standard
chemotherapy regimen as known in the art. Examples of such
chemotherapeutic agents include 5-fluorouracil, leucovorin, irinotecan,
gemcitabine-erlotinib, capecitabine and platinum-based chemotherapeutic
agents, such as paclitaxel, carboplatin, cisplatin and oxaliplatin. As
demonstrated in the appended examples, the addition of bevacizumab to
capecitabine- or 5-fluorouracil-based chemotherapeutic regimens effected
an increase in progression free survival and correlated with overall
survival in the gastric cancer patients and/or patient population defined
and selected according to the expression level of neuropilin, in
particular, having lower expression of neuropilin in tumor samples
relative to control levels established in similarly situated patients.

[0024] Bevacizumab may be combined with a capecitabine- or
5-fluorouracil-based chemotherapy regimen. The selection between
capecitabine and 5-fluorouracil is best determined by the treating
physician based on standards well established in the art. Examples of
capecitabine-based chemotherapy regimens include the combination of
capecitabine (or 5-fluorouracil) administered in combination with
cisplatin. A typical cycle of capecitabine/cisplatin therapy may be
capecitabine administered at a dose of 1000 mg/m2 orally twice daily
(bid) over days 1 to 14, followed by 1 week rest, and cisplatin at a dose
of 80 mg/m2 administered i.v. as a 2 hour infusion on day 1 of the
cycle with hyper-hydration and pre-medication (steroids and anti-emetics;
3×/week); the cisplatin and capecitabine cycle is continued until
disease progression or unmanageable toxicity, with cisplatin
administration limited to a maximum of 6 cycles. Accordingly, in certain
aspects of the invention, the patient identified according to the methods
herein is treated with bevacizumab in combination with
capecitabine/cisplatin. Common modes of administration of bevacizumab
include parenteral administration as a bolus dose or as an infusion over
a set period of time, e.g., administration of the total daily dose over
10 min., 20 min., 30 min., 40 min., 50 min., 60 min., 75 min., 90 min.,
105 min., 120 min., 3 hr., 4 hr., 5 hr. or 6 hr. For example, 7.5 mg/kg
of bevacizumab (Avastin®) may be administered to patients with
gastric cancer as an intravenous infusion over 15 to 30 minutes on day 1
of every capecitabine cycle as described above. The skilled person will
recognize that further modes of administration of bevacizumab are
encompassed by the invention as determined by the specific patient and
chemotherapy regimen, and that the specific mode of administration and
therapeutic dosage are best determined by the treating physician
according to methods known in the art.

[0025] The patients selected according to the methods of the present
invention are treated with bevacizumab in combination with a chemotherapy
regimen, and may be further treated with one or more additional
anti-cancer therapies. In certain aspects, the one or more additional
anti-cancer therapy is radiation.

[0026] In preferred embodiments, the sample obtained from the patient is
collected prior to beginning any other chemotherapeutic regimen or
therapy, e.g., therapy for the treatment of cancer or the management or
amelioration of a symptom thereof. Therefore, in preferred embodiments,
the sample is collected before the administration of chemotherapeutics or
the start of a chemotherapy regimen.

[0027] The present invention also relates to a diagnostic composition or
kit comprising oligonucleotides or polypeptides suitable for the
determination of the tumor specific expression level of neuropilin. As
detailed herein, oligonucleotides such as DNA, RNA or mixtures of DNA and
RNA probes may be of use in detecting mRNA levels of the marker/indicator
proteins, in particular, neuropilin, while polypeptides may be of use in
directly detecting protein levels of the marker/indicator proteins via
specific protein-protein interaction. In preferred aspects of the
invention, the polypeptides encompassed as probes for the expression
levels of neuropilin, and included in the kits or diagnostic compositions
described herein, are antibodies specific for neuropilin, or specific for
homologues, variants and/or truncations thereof.

[0028] Accordingly, a further embodiment of the present invention provides
a kit useful for carrying out the methods herein described, comprising
oligonucleotides or polypeptides capable of determining the expression
level of neuropilin. Preferably, the oligonucleotides comprise primers
and/or probes specific for the mRNA encoding neuropilin as defined and
described herein, and the polypeptides comprise proteins capable of
specific interaction with neuropilin, e.g., marker/indicator specific
antibodies or antibody fragments.

[0029] In a further embodiment, the present invention provides the use of
bevacizumab for improving treatment effect in a patient suffering from
gastric cancer, in particular, adenocarcinoma of the stomach or GEJ,
comprising the following steps:

[0030] (a) determining the expression
level of neuropilin in a patient sample; and

[0031] (b) administering
bevacizumab in combination with a chemotherapy regimen to the patient
having a decreased level of neuropilin relative to control levels
determined in patients suffering from gastric cancer, in particular,
adenocarcinoma of the stomach or GEJ. The improved treatment effect may
be improved overall survival or improved progression free survival.

[0032] As documented in the appended examples, the present invention
solves the identified technical problem in that it could surprisingly be
shown that the expression level of neuropilin in a given patient,
relative to a control level determined in patients diagnosed with gastric
cancer, in particular, adenocarcinoma of the stomach or GEJ, correlate
with treatment effect in patients administered bevacizumab in combination
with a capecitabine- or 5-fluorouracil-based chemotherapy regimen.

[0033] The phrase "responsive to" in the context of the present invention
indicates that a subject/patient suffering from, suspected to suffer or
prone to suffer from, or diagnosed with gastric cancer, in particular,
adenocarcinoma of the stomach or GEJ, shows a response to a chemotherapy
regimen comprising the addition of bevacizumab. A skilled person will
readily be in a position to determine whether a person treated with
bevacizumab according to the methods of the invention shows a response.
For example, a response may be reflected by decreased suffering from
gastric cancer, such as a diminished and/or halted tumor growth,
reduction of the size of a tumor, and/or amelioration of one or more
symptoms of gastric cancer, e.g., gastrointestinal bleeding, pain,
anemia. Preferably, the response may be reflected by decreased or
diminished indices of the metastatic conversion of gastric cancer, e.g.,
the prevention of the formation of metastases or a reduction of number or
size of metastases,

[0034] The phrase "sensitive to" in the context of the present invention
indicates that a subject/patient suffering from, suspected to suffer or
prone to suffer from, or diagnosed, with gastric cancer, in particular,
adenocarcinoma of the stomach or GEJ, shows in some way a positive
reaction to treatment with bevacizumab in combination with a chemotherapy
regimen. The reaction of the patient may be less pronounced when compared
to a patient "responsive to" as described hereinabove. For example, the
patient may experience less suffering associated with the disease, though
no reduction in tumor growth or metastatic indicator may be measured,
and/or the reaction of the patient to the bevacizumab in combination with
the chemotherapy regimen may be only of a transient nature, i.e., the
growth of (a) tumor and/or (a) metastasis(es) may only be temporarily
reduced or halted.

[0035] The phrase "a patient suffering from" in accordance with the
invention refers to a patient showing clinical signs of gastric cancer,
in particular, adenocarcinoma of the stomach or GEJ. The gastric cancer
may be metastatic, inoperable and/or locally advanced adenocarcinoma of
the stomach or gastro-esophageal junction ("GEJ"). The phrase "being
susceptible to" or "being prone to," in the context of gastric cancer,
refers to an indication disease in a patient based on, e.g., a possible
genetic predisposition, a pre- or eventual exposure to hazardous and/or
carcinogenic compounds, or exposure to carcinogenic physical hazards,
such as radiation.

[0036] The phrase "treatment effect" in the context of the present
invention encompasses the phrases "progression free survival" and
"overall survival".

[0037] The phrase "progression-free survival" in the context of the
present invention refers to the length of time during and after treatment
during which, according to the assessment of the treating physician or
investigator, the patient's disease does not become worse, i.e., does not
progress. As the skilled person will appreciate, a patient's
progression-free survival is improved or enhanced if the patient
experiences a longer length of time during which the disease does not
progress as compared to the average or mean progression free survival
time of a control group of similarly situated patients.

[0038] The phrase "overall survival" in the context of the present
invention refers to the average survival of the patient within a patient
group. As the skilled person will appreciate, a patient's overall
survival is improved or enhanced, if the patient belongs to a subgroup of
patients that has a statistically significant longer mean survival time
as compared to another subgroup of patients. Improved overall survival
may be evident in one or more subgroups of patients but not apparent when
the patient population is analysed as a whole.

[0039] The terms "administration" or "administering" as used herein mean
the administration of an angiogenesis inhibitor, e.g., bevacizumab
(Avastin®), and/or a pharmaceutical composition/treatment regimen
comprising an angiogenesis inhibitor, e.g., bevacizumab (Avastin®),
to a patient in need of such treatment or medical intervention by any
suitable means known in the art for administration of a therapeutic
antibody. Nonlimiting routes of administration include by oral,
intravenous, intraperitoneal, subcutaneous, intramuscular, topical,
intradermal, intranasal or intrabronchial administration (for example as
effected by inhalation). Particularly preferred in context of this
invention is parenteral administration, e.g., intravenous administration.
With respect to bevacizumab (Avastin®) for the treatment of
colorectal cancer, the preferred dosages according to the EMEA are 5
mg/kg or 10 mg/kg of body weight given once every 2 weeks or 7.5 mg/kg or
15 mg/kg of body weight given once every 3 weeks (for details see
http://www.emea.europa.eu/humandocs/PDFs/EPAR/avastin/emea-combined-h582e-
n.pdf).

[0040] The term "antibody" is herein used in the broadest sense and
includes, but is not limited to, monoclonal and polyclonal antibodies,
multispecific antibodies (e.g., bispecific antibodies), chimeric
antibodies, CDR grafted antibodies, humanized antibodies, camelized
antibodies, single chain antibodies and antibody fragments and fragment
constructs, e.g., F(ab')2 fragments, Fab-fragments, Fv-fragments,
single chain Fv-fragments (scFvs), bispecific scFvs, diabodies, single
domain antibodies (dAbs) and minibodies, which exhibit the desired
biological activity, in particular, specific binding to one or more of
VEGFA, HER2, neuropilin and CD31, or to homologues, variants, fragments
and/or isoforms thereof.

[0042] In the context of the present invention, "homology" with reference
to an amino acid sequence is understood to refer to a sequence identity
of at least 80%, particularly an identity of at least 85%, preferably at
least 90% and still more preferably at least 95% over the full length of
the sequence as defined by the SEQ ID NO(s) provided herein. In the
context of this invention, a skilled person would understand that
homology covers further allelic variation(s) of the marker/indicator
proteins in different populations and ethnic groups.

[0043] As used herein, the term "polypeptide" relates to a peptide, a
protein, an oligopeptide or a polypeptide which encompasses amino acid
chains of a given length, wherein the amino acid residues are linked by
covalent peptide bonds. However, peptidomimetics of such
proteins/polypeptides are also encompassed by the invention wherein amino
acid(s) and/or peptide bond(s) have been replaced by functional analogs,
e.g., an amino acid residue other than one of the 20 gene-encoded amino
acids, e.g., selenocysteine. Peptides, oligopeptides and proteins may be
termed polypeptides. The terms polypeptide and protein are used
interchangeably herein. The term polypeptide also refers to, and does not
exclude, modifications of the polypeptide, e.g., glycosylation,
acetylation, phosphorylation and the like. Such modifications are well
described in basic texts and in more detailed monographs, as well as in a
voluminous research literature.

[0044] The terms "treating" and "treatment" as used herein refer to
remediation of, improvement of, lessening of the severity of, or
reduction in the time course of the disease or any parameter or symptom
thereof. Preferably said patient is a human patient and the disease to be
treated is a gastric cancer, in particular, adenocarcinoma of the stomach
or GEJ. The terms "assessing" or "assessment" of such a patient relates
to methods of determining the expression levels of neuropilin, and/or for
selecting such patients based on the expression levels of such
marker/indicator proteins relative to control levels established in
patients diagnosed with metastatic colorectal cancer.

[0045] In addition to the methods described above, the invention also
encompasses further immunohistochemical methods for assessing the
expression level of neuropilin, such as by Western blotting and
ELISA-based detection. As is understood in the art, the expression level
of the marker/indicator proteins of the invention may also be assessed at
the mRNA level by any suitable method known in the art, such as Northern
blotting, real time PCR, and RT PCR. Immunohistochemical- and mRNA-based
detection methods and systems are well known in the art and can be
deduced from standard textbooks, such as Lottspeich (Bioanalytik,
Spektrum Akademisher Verlag, 1998) or Sambrook and Russell (Molecular
Cloning: A Laboratory Manual, CSH Press, Cold Spring Harbor, N.Y.,
U.S.A., 2001). The described methods are of particular use for
determining the expression level, e.g., tumor specific expression level,
of neuropilin in a patient or group of patients relative to control
levels established in a similarly situated population, e.g., suffering
from or diagnosed with gastric cancer, in particular, adenocarcinoma of
the stomach or GEJ.

[0046] The expression level of neuropilin can also be determined on the
protein level by taking advantage of immunoagglutination,
immunoprecipitation (e.g., immunodiffusion, immunelectrophoresis, immune
fixation), western blotting techniques (e.g., (in situ) immuno
histochemistry, (in situ) immuno cytochemistry, affinity chromatography,
enzyme immunoassays), and the like. Amounts of purified polypeptide in
solution may also be determined by physical methods, e.g. photometry.
Methods of quantifying a particular polypeptide in a mixture usually rely
on specific binding, e.g., of antibodies. Specific detection and
quantitation methods exploiting the specificity of antibodies comprise
for example immunohistochemistry (in situ). For example, the
concentration/amount of the marker/indicator proteins of the present
invention (e.g., NRP-1, NRP-2 and/or a variant, homolog or truncation
thereof) in a cell or tissue may be determined by enzyme
linked-immunosorbent assay (ELISA). Alternatively, Western Blot analysis
or immunohistochemical staining can be performed. Western blotting
combines separation of a mixture of proteins by electrophoresis and
specific detection with antibodies. Electrophoresis may be
multi-dimensional such as 2D electrophoresis. Usually, polypeptides are
separated in 2D electrophoresis by their apparent molecular weight along
one dimension and by their isoelectric point along the other direction.

[0047] As mentioned above, the decreased expression of the
marker/indicator proteins according to the present invention may also be
reflected in a decreased expression of the corresponding gene(s) for
neuropilin (as described and defined herein). Therefore, a quantitative
assessment of the gene product prior to translation (e.g. spliced,
unspliced or partially spliced mRNA) can be performed in order to
evaluate the expression of the corresponding gene(s). The person skilled
in the art is aware of standard methods to be used in this context or may
deduce these methods from standard textbooks (e.g. Sambrook, 2001, loc.
cit.). For example, quantitative data on the respective
concentration/amounts of mRNA encoding neuropilin can be obtained by
Northern Blot, Real Time PCR and the like.

[0048] In a further aspect of the invention, the kit of the invention may
advantageously be used for carrying out a method of the invention and
could be, inter alia, employed in a variety of applications, e.g., in the
diagnostic field or as a research tool. The parts of the kit of the
invention can be packaged individually in vials or in combination in
containers or multicontainer units. Manufacture of the kit follows
preferably standard procedures which are known to the person skilled in
the art. The kit or diagnostic compositions may be used for detection of
the expression level of neuropilin (as defined and described herein) in
accordance with the herein-described methods of the invention, employing,
for example, immunohistochemical techniques.

[0049] Although exemplified by the use of bevacizumab, the invention
encompasses the use of other angiogenesis inhibitors known in the art for
use in combination with standard chemotherapy regimens. The terms
"angiogenesis inhibitor" as used herein refers to all agents that alter
angiogenesis (e.g. the process of forming blood vessels) and includes
agents that block the formation of and/or halt or slow the growth of
blood vessels. Nonlimiting examples of angiogenesis inhibitors include,
in addition to bevacizumab, pegaptanib, sunitinib, sorafenib and
vatalanib. Preferably, the angiogenesis inhibitor for use in accordance
with the methods of the present invention is bevacizumab. As used herein,
the term "bevacizumab" encompass all corresponding anti-VEGF antibodies
or anti-VEGF antibody fragments, that fulfil the requirements necessary
for obtaining a marketing authorization as an identical or biosimilar
product in a country or territory selected from the group of countries
consisting of the USA, Europe and Japan.

[0050] For use in the detection methods described herein, the skilled
person has the ability to label the polypeptides or oligonucleotides
encompassed by the present invention. As routinely practiced in the art,
hybridization probes for use in detecting mRNA levels and/or antibodies
or antibody fragments for use in IHC methods can be labelled and
visualized according to standard methods known in the art. Nonlimiting
examples of commonly used systems include the use of radiolabels, enzyme
labels, fluorescent tags, biotin-avidin complexes, chemiluminescence, and
the like.

[0051] The person skilled in the art, for example, the attending
physician, is readily in a position to administer the bevacizumab in
combination with a chemotherapy regimen to the patient/patient group as
selected and defined herein. In certain contexts, the attending physician
may modify, change or amend the administration schemes for the
bevacizumab and the chemotherapy regimen in accordance with his/her
professional experience. Therefore, in certain aspects of the present
invention, a method is provided for the treatment or improving treatment
effect (i.e., the progression-free or overall survival) in a patient
suffering from or suspected to suffer from gastric cancer with
bevacizumab in combination with a chemotherapy regimen, whereby said
patient/patient group is characterized in the assessment of a biological
sample (in particular a gastric tissue resection, gastric tissue biopsy
and/or metastatic lesion), said sample exhibiting a decreased expression
level of neuropilin, relative to control levels established in patients
suffering from and/or diagnosed with gastric cancer, in particular,
adenocarcinoma of the stomach or GEJ. The present invention also provides
for the use of bevacizumab in the preparation of pharmaceutical
composition for the treatment of a patient suffering from or suspected to
suffer from gastric cancer, in particular, adenocarcinoma of the stomach
or GEJ, wherein the patients are selected or characterized by the herein
disclosed protein marker/indicator status (i.e., a decreased expression
level of neuropilin relative to control levels established in patients
suffering from gastric cancer, in particular, adenocarcinoma of the
stomach or GEJ).

[0056] FIG. 4: Correlation between neuropilin expression with overall
survival, time to progression or death, and overall response rate (ORR).

EXAMPLES

[0057] Tissue samples were collected from patients participating a
randomized phase-III study comparing the results of adding bevacizumab to
first-line capecitabine (5-fluoruracil was allowed if capecitabine was
contraindicated)/cisplatin combination chemotherapy regimens for the
treatment of metastatic or inoperable, locally advanced adenocarcinoma of
the stomach or GEJ (the AVAGAST study, see, Kang et al., 2010, J. Clin.
Oncol., 28:18s (suppl. abstr. LBA4007) ("Kang")). An investigation of the
status of biomarkers related to angiogenesis and tumorigenesis revealed
that a decreased expression level of neuropilin relative to a control
level determined in the entire patient population indicated improved
overall survival and/or progression free survival.

[0058] Patients and Immunohistochemical Methods

[0059] A total of 774 patients participated in the AVAGAST study, and
tumor samples from between 629 and 727 of the participants were available
for biomarker analysis, dependent on the specific biomarker. Treatment
arms were balanced. Approximately 95% of the patients were metastatic.
Approximately 2/3 of the patients were male, 49% were from Asia/Pacific,
32% were from Europe and 19% were from the Americas (see, Kang).

[0060] Tissue samples were available as tissue blocks or as previously
prepared slides. Immunohistochemical analysis was performed on 5 μm
sections of formalin-fixed paraffin-embedded tissue samples (for blocks)
or on the previously prepared slides. After deparaffinization and
rehydration, antigen retrieval was performed by citrate pH 6.0 buffer at
95° C. for 30 minutes in a PT module or CC1 buffer in the
Benchmark-XT (Ventana, Tucson, Ariz., USA).

[0061] Initial biomarkers, including neuropilin, were selected for
immunohistochemical analysis based on known tumorigenic and angiogenic
activity. In particular, neuropilin was analysed using the anti-human
neuropilin murine monoclonal antibody available from Santa Cruz
Biotechnology, Inc. (Santa Cruz, Calif., U.S.A) as catalog number
sc-5307.

[0062] Sections were stained on Autostainer or Benchmark-XT (for VEGFR-1)
and primary antibodies were incubated for 1 hour. With specific respect
to the Santa Cruz antibody, this anti-neuropilin antibody was used at
1/50 dilution. Binding of the primary antibodies was visualized using the
Envision system (DAKO, Glostrup, Denmark) or Ultraview (Ventana, Tucson,
Ariz. USA). All sections were counterstained with Mayer's hematoxylin.

[0063] Validation reports showing accuracy, specificity, linearity, and
precision (reproducibility and repeatability) were produced for each IHC
assay. Staining of external control slides and intrinsic control elements
was documented.

[0064] Statistical Analysis

[0065] The overall distribution of biomarkers was described using the
H-score for tumor markers. The number of markers examined was limited and
each one was supported by a biological rationale; there was no formal
correction for multiple testing. The a priori cut-off was used for
protein expression level: median (below, above) and quartile (≦25,
25<x≦50, 50<x≦75, >75).

[0066] Treatment effects were estimated in subgroups of patients defined
by biomarker level. Overall survival ("OS") and/or progression free
survival ("PFS") was chosen as the primary endpoint; the primary
descriptive analysis was performed using subgroup analysis. Test of
treatment by biomarker interactions (median cut-off) also provided a
secondary analysis.

[0067] Results

[0068] Tumor Markers

[0069] Results of the analysis of the tumor samples for neuropilin are
provided in Table 1.

[0073] The calculated hazard ratios indicate that overall survival
improves in those patients exhibiting relatively decreased tumor specific
expression of neuropilin when administered bevacizumab in combination
with the standard chemotherapy. In particular, in Table 2, the upper
bound of the 95% confidence interval of treatment hazard ratio in the
subset of patients with low tumor specific neuropilin expression
(≦median) is below 1. This supports the statistical relevance of
the treatment effect (overall survival) observed in this sub-group of
patients.

[0074] A Kaplan-Meier curve correlating bevacizumab treatment and
neuropilin expression with respect to overall survival is provided in
FIG. 1 (median cut-off). The improvement in overall survival for those
patients having relatively low neuropilin expression when bevacizumab is
added to the chemotherapy, indicated in the hazard ratios, is also
visible in FIG. 5. Median overall survival was improved by 1.8 months in
patients with relatively low tumor specific neuropilin expression
(≦median) compared to only 0.8 months for patients with tumor
specific neuropilin expression above median. The results demonstrate that
the treatment effect (overall survival) is improved in the subset of
patients with relatively low level of neuropilin.

[0075] Biomarker Correlation with Progression Free Survival

[0076] Hazard ratios were determined for time to disease progression or
death in patients separated by median or quartile neuropilin H scores.

[0077] The calculated hazard ratios indicate that progression free
survival improves in those patients administered bevacizumab in
combination with the standard chemotherapy as the tumor specific
expression of neuropilin decreases. In Table 4, the upper bound of the
95% confidence interval of treatment hazard ratio in the subset of
patients with low tumor specific neuropilin expression (≦median)
is below 1. This supports the statistical relevance of the treatment
effect (progression free survival) observed in this sub-group of
patients.

[0078] Table 5 was produced in the per-protocol population which excluded
patients with major protocol violations. Table 6 was produced in the
intent-to-treat population which included all randomized patients. Table
6, therefore, provides a more accurate analysis.

[0079] A Kaplan-Meier curve correlating bevacizumab treatment and
neuropilin expression with respect to progression free survival is
provided in FIG. 2 (median cut-off). The improvement in progression free
survival for those patients having relatively low neuropilin expression
when bevacizumab is added to the chemotherapy, indicated in the hazard
ratios, is also visible in FIG. 2. Median progression free survival was
improved by 2.1 months in patients with relatively low tumor specific
neuropilin expression (≦median) compared to only 1.3 months for
patients with tumor specific neuropilin expression above median. The
results demonstrate that the treatment effect (progression free survival)
is improved in the subset of patients with relatively low level of
neuropilin.